Human S-adenosyl-L-methionine methyltransferase

ABSTRACT

The invention provides a human S-adenosyl-L-methionine methyltransferase (SAM-MT) and polynucleotides which identify and encode SAM-MT. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of SAM-MT.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/149,534, filed Sep. 8, 1998, which is a divisionalapplication of U.S. application Ser. No. 08/900,565, filed Jul. 25,1997, now U.S. Pat. No. 5,876,996, issued Mar. 2, 1999, both entitledHUMAN S-ADENOSYL-L-METHIONINE METHYLTRANSFERASE, all of whichapplications and patents are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a human S-adenosyl-L-methyltransferase and to the use of thesesequences in the diagnosis, prevention, and treatment of neoplastic,immunological, and vesicle trafficking disorders.

BACKGROUND OF THE INVENTION

[0003] Covalent modification of cellular substrates with methyl groupshas been implicated in the pathology of cancer and other diseases(Gloria, L. et al. (1996) Cancer 78:2300-2306). Cytosinehypermethylation of eukaryotic DNA prevents transcriptional activation(Turker, M. S. and Bestor, T. H. (1997) Mutat. Res. 386:119-130).N⁶-methyladenosine is found at internal positions of mRNA in highereukaryotes (Bokar, J. A. et al. (1994) J. Biol. Chem. 269:17697-17704).Hypermethylated viral DNA is transcribed at higher rates than hypo- orhemimethylated DNA in infected cells (Willis, D. B. et al. (1989) Cell.Biophys. 15:97-111).

[0004] Many pathways of small molecule degradation, such as those ofneurotransmitters, require methyltransferase activity (Kagan, R. M. andClarke, S. (1994) Arch. Biochem. Biophys. 310:417-427). Degradation ofcatecholamines (epinephrine or norepinephrine) requiresphenylethanolamine -methyltransferase. Hydroxyindole methyltransferaseconverts N-acetyl-5-hydroxytryptamine to melatonin in the pineal gland.

[0005] S-adenosylmethionine (AdoMet) is an important source of methylgroups for methylation reactions in the cell (Bottiglieri, T. andHyland, K. (1994) Acta Neurol. Scand. Suppl. 154:19-26).Methyltransferase activity catalyzes the transfer of methyl groups fromAdoMet to acceptor molecules such as phosphotidylethanolamine or thepolynucleotide 5′ cap of viral mRNA (Montgomery, J. A. et al. (1982) J.Med. Chem. 25:626-629).

[0006] Members of the S-adenosylmethionine methyltransferase family(AdoMet-MT), utilize AdoMet as a substrate or product and harbor threecommon consensus sequence motifs. Motifs I and II are characteristicallyspaced between 34 and 90 (mode 52, median 52-54) amino acid residuesapart; motifs II and III are spaced between 12 and 38 (mode 22, median20-22) residues apart. Motif I comprises part of the AdoMet bindingpocket; motif II may also be involved in binding AdoMet; the role ofmotif III is uncertain (Kagan, R. M. and Clarke, S. (supra)).

[0007] Messenger RNA N⁶-adenosine methyltransferase holoenzyme has beenpartially purified from HeLa cell nuclear extract to yield threesubunits, an 875 kDa ssDNA-agarose binding protein, a 70 kDaAdoMet-binding protein, and an approximately 30 kDa component withunknown function. The three components are absolutely required for RNAm⁶A-methylation activity (Bokar, J. A. (supra)).

[0008] The nematode Caenorhabditis elegans employs many of the samemethyltransferase activities found in higher animals (Kagan, R. M. andClarke, S. (1995) Biochemistry, 34:10794-10806). A C. elegans C27F2 geneproduct identified as a member of the methyltransferase family has nowbeen described (Wilson, R. et al. (1994) Nature 368:32-38).

[0009] In their roles as a rate-limiting step in methyltransferasereactions, AdoMet-MTs have been identified as a target for psychiatric,antiviral, anticancer and anti-inflammatory drug design (Bottiglieri, T.and Hyland, K. (supra); Gloria, L. et al. (supra)). Sequence-specificmethylation inhibits the activity of the Epstein-Barr virus LMP 1 andBCR2 enhancer-promoter regions (Minarovits, J. et al. (1994) Virology200:661-667). 2′-5′-linked oligo(adenylic acid) nucleoside analoguessynthesized by interferon-treated mouse L cells act as antiviral agents(Goswami, B. B. et al, (1982) J. Biol. Chem. 257:6867-6870). Adenineanalogue inhibitors of AdoMet-MT decreased nucleic acid methylation andproliferation of leukemia L1210 cells (Kramer, D. L. et al. (1990)Cancer Res. 50:3838-3842).

[0010] The discovery of a new human S-adenosyl-L-methioninemethyltransferase and the polynucleotides encoding it satisfies a needin the art by providing new compositions which are useful in thediagnosis, prevention and treatment of neoplastic, immunological, andvesicle trafficking disorders.

SUMMARY OF THE INVENTION

[0011] The invention features a substantially purified polypeptide,human S-adenosyl-L-methionine methyltransferase (SAM-MT), having theamino acid sequence shown in SEQ ID NO:1, or fragments thereof.

[0012] The invention further provides an isolated and substantiallypurified polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or fragments thereof and acomposition comprising said polynucleotide sequence. The invention alsoprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence encoding the amino acidsequence SEQ ID NO:1, or fragments of said polynucleotide sequence. Theinvention further provides a polynucleotide sequence comprising thecomplement of the polynucleotide sequence encoding the amino acidsequence of SEQ ID NO:1, or fragments or variants of said polynucleotidesequence.

[0013] The invention also provides an isolated and purified sequencecomprising SEQ ID NO:2 or variants thereof. In addition, the inventionprovides a polynucleotide sequence which hybridizes under stringentconditions to the polynucleotide sequence of SEQ ID NO:2. The inventionalso provides a polynucleotide sequence comprising the complement of SEQID NO:2, or fragments or variants thereof.

[0014] The present invention further provides an expression vectorcontaining at least a fragment of any of the claimed polynucleotidesequences. In yet another aspect, the expression vector containing thepolynucleotide sequence is contained within a host cell.

[0015] The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding SAM-MT under conditions suitable forthe expression of the polypeptide; and b) recovering the polypeptidefrom the host cell culture.

[0016] The invention also provides a pharmaceutical compositioncomprising a substantially purified SAM-MT having the amino acidsequence of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

[0017] The invention also provides a purified antagonist of thepolypeptide of SEQ ID NO:1. In one aspect the invention provides apurified antibody which binds to a polypeptide comprising the amino acidsequence of SEQ ID NO:1.

[0018] Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

[0019] The invention also provides a method for treating or preventing aneoplastic disorder comprising administering to a subject in need ofsuch treatment an effective amount of a purified antagonist to SAM-MT.

[0020] The invention also provides a method for treating or preventingan immunological disorder comprising administering to a subject in needof such treatment an effective amount of a purified antagonist toSAM-MT.

[0021] The invention also provides a method for treating or preventing avesicle trafficking disorder comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising purified SAM-MT.

[0022] The invention also provides a method for detecting apolynucleotide which encodes SAM-MT in a biological sample comprisingthe steps of: a) hybridizing the complement of the polynucleotidesequence which encodes SEQ ID NO:1 to nucleic acid material of abiological sample, thereby forming a hybridization complex; and b)detecting the hybridization complex, wherein the presence of the complexcorrelates with the presence of a polynucleotide encoding SAM-MT in thebiological sample. In one aspect the nucleic acid material of thebiological sample is amplified by the polymerase chain reaction prior tohybridization.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of SAM-MT. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

[0024]FIGS. 2A and 2B show the amino acid sequence alignments amongSAM-MT (10625; SEQ ID NO:1), Caenorhabditis elegans putativemethyltransferase (GI 1065505; SEQ ID NO:3) and Saccharomyces cerevisiaeputative methyltransferase (GI 1907189; SEQ ID NO:4), produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc,Madison Wis.).

[0025]FIGS. 3A and 3B show the hydrophobicity plots for SAM-MT, SEQ IDNO:1 and Caenorhabditis elegans putative methyltransferase (SEQ IDNO:3), respectively; the positive X axis reflects amino acid position,and the negative Y axis, hydrophobicity (MACDNASIS PRO software).

[0026]FIGS. 4A and 4B show the amino acid sequence alignments betweenSAM-MT (10625; SEQ ID NO:1) and the common consensus sequence motifs,motifs I, II, and III (AdoMet-MT) of enzymes that utilize AdoMet as asubstrate or product, produced using the multisequence alignment programof DNASTAR software (DNASTAR Inc, Madison Wis.).

DESCRIPTION OF THE INVENTION

[0027] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0028] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0029] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

[0030] SAM-MT, as used herein, refers to the amino acid sequences ofsubstantially purified SAM-MT obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0031] The term “agonist”, as used herein, refers to a molecule which,when bound to SAM-MT, increases or prolongs the duration of the effectof SAM-MT. Agonists may include proteins, nucleic acids, carbohydrates,or any other molecules which bind to and modulate the effect of SAM-MT.

[0032] An “allele” or “allelic sequence”, as used herein, is analternative form of the gene encoding SAM-MT. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0033] “Altered” nucleic acid sequences encoding SAM-MT, as used herein,include those with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent SAM-MT. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding SAM-MT,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding SAM-MT. The encoded protein may also be “altered” and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent SAM-MT.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of SAM-MT is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

[0034] “Amino acid sequence”, as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of SAM-MT arepreferably about 5 to about 15 amino acids in length and retain thebiological activity or the immunological activity of SAM-MT. Where“amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, amino acid sequence,and like terms, are not meant to limit the amino acid sequence to thecomplete, native amino acid sequence associated with the recited proteinmolecule.

[0035] “Amplification”, as used herein, refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0036] The term “antagonist”, as used herein, refers to a moleculewhich, when bound to SAM-MT, decreases the amount or the duration of theeffect of the biological or immunological activity of SAM-MT.Antagonists may include proteins, nucleic acids, carbohydrates,antibodies or any other molecules which decrease the effect of SAM-MT.

[0037] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind SAM-MTpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or oligopeptide used to immunize an animal can be derivedfrom the translation of RNA or synthesized chemically and can beconjugated to a carrier protein, if desired. Commonly used carriers thatare chemically coupled to peptides include bovine serum albumin andthyroglobulin, keyhole limpet hemocyanin. The coupled peptide is thenused to immunize the animal (e.g., a mouse, a rat, or a rabbit).

[0038] The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0039] The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

[0040] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic SAM-MT, orany oligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0041] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

[0042] A “composition comprising a given polynucleotide sequence”, asused herein, refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding SAM-MT (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2and fragments thereof) may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0043] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, has been extendedusing XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction and resequenced, or has been assembled from the overlappingsequences of more than one Incyte Clone using a computer program forfragment assembly (e.g., GELVIEW fragment assembly system, GCG, Madison,Wis.). Some sequences have been both extended and assembled to producethe consensus sequence.

[0044] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding SAM-MT in a sample and therebycorrelates with expression of the transcript from the polynucleotideencoding the protein.

[0045] A “deletion”, as used herein, refers to a change in the aminoacid or nucleotide sequence and results in the absence of one or moreamino acid residues or nucleotides.

[0046] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to SAM-MT orthe encoded SAM-MT. Such modifications include, for example, replacementof hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

[0047] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

[0048] Human artificial chromosomes (HACs) are linear microchromosomeswhich may contain DNA sequences of 10K to 10M in size and contain all ofthe elements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

[0049] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0050] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0051] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

[0052] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

[0053] “Microarray” refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

[0054] The term “modulate”, as used herein, refers to a change in theactivity of SAM-MT. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional or immunological properties of SAM-MT.

[0055] “Nucleic acid sequence”, as used herein, refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments thereof,and to DNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand.“Fragments” are those nucleic acid sequences which are greater than 60nucleotides in length, and most preferably includes fragments that areat least 100 nucleotides or at least 1000 nucleotides, and at least10,000 nucleotides in length.

[0056] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to about 60 nucleotides, preferably about15 to 30 nucleotides, and more preferably about 20 to 25 nucleotides,which can be used in PCR amplification or a hybridization assay, or amicroarray. As used herein, oligonucleotide is substantially equivalentto the terms “amplimers”,“primers”, “oligomers”, and “probes”, ascommonly defined in the art.

[0057] “Peptide nucleic acid”, PNA, as used herein, refers to anantisense molecule or anti-gene agent which comprises an oligonucleotideof at least five nucleotides in length linked to a peptide backbone ofamino acid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

[0058] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from five amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length SAM-MT and fragments thereof.

[0059] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encodingSAM-MT, or fragments thereof, or SAM-MT itself may comprise a bodilyfluid, extract from a cell, chromosome, organelle, or membrane isolatedfrom a cell, a cell, genomic DNA, RNA, or cDNA (in solution or bound toa solid support, a tissue, a tissue print, and the like).

[0060] The terms “specific binding” or “specifically binding”, as usedherein, refer to that interaction between a protein or peptide and anagonist, an antibody and an antagonist. The interaction is dependentupon the presence of a particular structure (i.e., the antigenicdeterminant or epitope) of the protein recognized by the bindingmolecule. For example, if an antibody is specific for epitope “A”, thepresence of a protein containing epitope A (or free, unlabeled A) in areaction containing labeled “A” and the antibody will reduce the amountof labeled A bound to the antibody.

[0061] The terms “stringent conditions” or “stringency”, as used herein,refer to the conditions for hybridization as defined by the nucleicacid, salt, and temperature. These conditions are well known in the artand may be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

[0062] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0063] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0064] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0065] A “variant” of SAM-MT, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNASTAR software.

The Invention

[0066] The invention is based on the discovery of a new humanS-adenosyl-L-methionine methyltransferase (hereinafter referred to as“SAM-MT”), the polynucleotides encoding SAM-MT, and the use of thesecompositions for the diagnosis, prevention, or treatment of neoplastic,immunological, and vesicle trafficking disorders.

[0067] Nucleic acids encoding the SAM-MT of the present invention werefirst identified in Incyte Clone 10625 from the THP-1 promonocyte cellline, PMA+LPS stimulated, cDNA library (THP1PLB01) using a computersearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the following overlapping and/or extended nucleicacid sequences: Incyte Clones 10625 (THP1PLB01), 1749286 (STOMTUT02),1689223 (PROSTUT10), 075978 (THP1PEB01), and 2731022 (OVARTUT04).

[0068] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, and 1C. SAM-MT is 281 amino acids in length, with a predictedrelative molecular mass of 31.9 kDa (MACDNASIS PRO software). SAM-MT hasthree potential protein kinase C phosphorylation sites at residuesS-194, S-240, and T-273, and one potential tyrosine kinasephosphorylation site at residue Y-48. As shown in FIGS. 2A and 2B,SAM-MT has chemical and structural homology with the putativemethyltransferases from C. elegans (GI 1065505; SEQ ID NO:3) and S.cerevisiae (GI 1907189; SEQ ID NO:4). In particular, SAM-MT and C.elegans putative methyltransferase share 51 % amino acid sequenceidentity, share the AdoMet-MT motifs I and III and share one proteinkinase C phosphorylation site. As illustrated by FIGS. 3A and 3B ,SAM-MT and C. elegans putative methyltransferase have rather similarhydrophobicity plots.

[0069] As shown in FIGS. 4A and 4B, SAM-MT contains three commonconsensus sequence motifs of the small molecule methyltransferaseenzymes (AdoMet-MT) that utilize AdoMet as a substrate or product.

[0070] Northern analysis shows the expression of this sequence invarious libraries, at least 60% of which are immortalized or cancerous,50% are from secretory tissue, and at least 41% of which involve immuneresponse. Of particular note is the expression of SAM-MT in gut,reproductive, and neural tissue; in proliferating cells; in fetal lung,gut, and heart; and in placenta.

[0071] The invention also encompasses SAM-MT variants. A preferredSAM-MT variant is one having at least 80%, and more preferably at least90%, amino acid sequence identity to the SAM-MT amino acid sequence (SEQID NO:1) and which retains at least one biological, immunological orother functional characteristic or activity of SAM-MT. A most preferredSAM-MT variant is one having at least 95% amino acid sequence identityto SEQ ID NO:1.

[0072] The invention also encompasses polynucleotides which encodeSAM-MT. Accordingly, any nucleic acid sequence which encodes the aminoacid sequence of SAM-MT can be used to produce recombinant moleculeswhich express SAM-MT. In a particular embodiment, the inventionencompasses the polynucleotide comprising the nucleic acid sequence ofSEQ ID NO:2 as shown in FIGS. 1A, 1B, and 1C.

[0073] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding SAM-MT, some bearing minimal homology to thenucleotide sequences of any known and naturally occurring gene, may beproduced. Thus, the invention contemplates each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring SAM-MT, and all suchvariations are to be considered as being specifically disclosed.

[0074] Although nucleotide sequences which encode SAM-MT and itsvariants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring SAM-MT under appropriately selectedconditions of stringency, it may be advantageous to produce nucleotidesequences encoding SAM-MT or its derivatives possessing a substantiallydifferent codon usage. Codons may be selected to increase the rate atwhich expression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding SAM-MT and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0075] The invention also encompasses production of DNA sequences, orfragments thereof, which encode SAM-MT and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art. Moreover,synthetic chemistry may be used to introduce mutations into a sequenceencoding SAM-MT or any fragment thereof.

[0076] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

[0077] Methods for DNA sequencing which are well known and generallyavailable in the art and may be used to practice any of the embodimentsof the invention. The methods may employ such enzymes as the Klenowfragment of DNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland,Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase(Amersham, Chicago, Ill.), or combinations of polymerases andproofreading exonucleases such as those found in the ELONGASEAmplification System marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the process is automated with machines such as the HamiltonMicro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI Catalyst and 373 and 377 DNASequencers (Perkin Elmer).

[0078] The nucleic acid sequences encoding SAM-MT may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

[0079] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

[0080] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (199 1) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

[0081] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0082] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

[0083] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0084] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode SAM-MT may be used in recombinant DNAmolecules to direct expression of SAM-MT, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressSAM-MT.

[0085] As will be understood by those of skill in the art, it may beadvantageous to produce SAM-MT-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

[0086] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterSAM-MT encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0087] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding SAM-MT may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of SAM-MT activity, it may be useful toencode a chimeric SAM-MT protein that can be recognized by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the SAM-MT encoding sequenceand the heterologous protein sequence, so that SAM-MT may be cleaved andpurified away from the heterologous moiety.

[0088] In another embodiment, sequences encoding SAM-MT may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of SAM-MT, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

[0089] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of SAM-MT, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0090] In order to express a biologically active SAM-MT, the nucleotidesequences encoding SAM-MT or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0091] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encodingSAM-MT and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook, J. et al. (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y.

[0092] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding SAM-MT. These include, but arenot limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems. The invention is not limited by the host cell employed.

[0093] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding SAM-MT,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0094] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for SAM-MT. For example, whenlarge quantities of SAM-MT are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding SAM-MT may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0095] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0096] In cases where plant expression vectors are used, the expressionof sequences encoding SAM-MT may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0097] An insect system may also be used to express SAM-MT. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encodingSAM-MT may be cloned into a non-essential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of SAM-MT will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which SAM-MT may be expressed(Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0098] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding SAM-MT may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing SAM-MT in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0099] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

[0100] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding SAM-MT. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding SAM-MT, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0101] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0102] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress SAM-MT may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0103] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0104] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding SAM-MTis inserted within a marker gene sequence, transformed cells containingsequences encoding SAM-MT can be identified by the absence of markergene function. Alternatively, a marker gene can be placed in tandem witha sequence encoding SAM-MT under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0105] Alternatively, host cells which contain the nucleic acid sequenceencoding SAM-MT and express SAM-MT may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0106] The presence of polynucleotide sequences encoding SAM-MT can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments or fragments of polynucleotides encoding SAM-MT.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding SAM-MT todetect transformants containing DNA or RNA encoding SAM-MT.

[0107] A variety of protocols for detecting and measuring the expressionof SAM-MT, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson SAM-MT is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0108] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encodingSAM-MT include oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding SAM-MT, or any fragments thereof may be cloned into a vectorfor the production of an mRNA probe. Such vectors are known in the art,are commercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used forease of detection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

[0109] Host cells transformed with nucleotide sequences encoding SAM-MTmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode SAM-MT may be designed to contain signal sequences which directsecretion of SAM-MT through a prokaryotic or eukaryotic cell membrane.Other constructions may be used to join sequences encoding SAM-MT tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and SAM-MT may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingSAM-MT and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying SAM-MT from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0110] In addition to recombinant production, fragments of SAM-MT may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of SAM-MT may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

Therapeutics

[0111] Chemical and structural homology exists among SAM-MT and putativemethyltransferases from C. elegans (GI 1065505) and S. cerevisiae (GI1907189). In addition, SAM-MT is expressed in tumors; in gut,reproductive, and neural tissue; in proliferating cells; in secretorycells, in fetal lung, gut, and heart; and in placenta. Therefore, SAM-MTappears to play a role in neoplastic, immunological, and vesicletrafficking disorders where SAM-MT is overexpressed.

[0112] Therefore, in one embodiment, an antagonist of SAM-MT may beadministered to a subject to prevent or treat a neoplastic disorder.Such disorders may include, but are not limited to, adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, andparticularly cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. In one aspect, an antibody which specifically binds SAM-MT maybe used directly as an antagonist or indirectly as a targeting ordelivery mechanism for bringing a pharmaceutical agent to cells ortissue which express SAM-MT.

[0113] In another embodiment, a vector expressing the complement of thepolynucleotide encoding SAM-MT may be administered to a subject to treator prevent a neoplastic disorder including, but not limited to, thosedescribed above.

[0114] In one embodiment, an antagonist of SAM-MT may be administered toa subject to treat an immunological disorder. Such disorders mayinclude, but are not limited to AIDS, Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjören's syndrome,Werner syndrome, and autoimmune thyroiditis; complications of cancer,hemodialysis, extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections and trauma. In oneaspect, an antibody which specifically binds SAM-MT may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express SAM-MT.

[0115] In another embodiment, a vector expressing the complement of thepolynucleotide encoding SAM-MT may be administered to a subject to treator prevent an immunological disorder including, but not limited to,those described above.

[0116] In one embodiment, SAM-MT or a fragment or derivative thereof maybe administered to a subject to treat a disorder associated with vesicletrafficking. Such disorders include, but are not limited to, cysticfibrosis, glucose-galactose malabsorption syndrome,hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- andhypoglycemia, Grave's disease, goiter, Cushing's disease, Addison'sdisease; gastrointestinal disorders including ulcerative colitis,gastric and duodenal ulcers; and other conditions associated withabnormal vesicle trafficking including AIDS; and allergies including hayfever, asthma, and urticaria (hives); autoimmune hemolytic anemia;proliferative glomerulonephritis; inflammatory bowel disease; multiplesclerosis; myasthenia gravis; rheumatoid and osteoarthritis;scleroderma; Chediak-Higashi and Sjören's syndromes; systemic lupuserythematosus; toxic shock syndrome; traumatic tissue damage; and viral,bacterial, fungal, helminth, and protozoal infections.

[0117] In another embodiment, a vector capable of expressing SAM-MT, ora fragment or a derivative thereof, may also be administered to asubject to treat a disorder associated with vesicle traffickingincluding, but not limited to, those listed above.

[0118] In still another embodiment, an agonist of SAM-MT may also beadministered to a subject to treat a disorder associated with vesicletrafficking including, but not limited to, those listed above.

[0119] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0120] An antagonist of SAM-MT may be produced using methods which aregenerally known in the art, In particular, purified SAM-MT may be usedto produce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind SAM-MT.

[0121] Antibodies to SAM-MT may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0122] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith SAM-MT or any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0123] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to SAM-MT have an amino acid sequenceconsisting of at least five amino acids and more preferably at least 10amino acids. It is also preferable that they are identical to a portionof the amino acid sequence of the natural protein, and they may containthe entire amino acid sequence of a small, naturally occurring molecule.Short stretches of SAM-MT amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0124] Monoclonal antibodies to SAM-MT may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0125] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceSAM-MT-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Kang, A. S. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0126] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0127] Antibody fragments which contain specific binding sites forSAM-MT may also be generated. For example, such fragments include, butare not limited to, the F(ab′)2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)2fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0128] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between SAM-MT and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering SAM-MT epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0129] In another embodiment of the invention, the polynucleotidesencoding SAM-MT, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding SAM-MT may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding SAM-MT. Thus, complementary molecules orfragments may be used to modulate SAM-MT activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding SAM-MT.

[0130] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encodingSAM-MT. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

[0131] Genes encoding SAM-MT can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes SAM-MT. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0132] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′ or regulatory regions of the geneencoding SAM-MT (signal sequence, promoters, enhancers, and introns).Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0133] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding SAM-MT.

[0134] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0135] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding SAM-MT. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA constitutively or inducibly can be introduced into cell lines,cells, or tissues.

[0136] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0137] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposome injectionsor polycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

[0138] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0139] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of SAM-MT,antibodies to SAM-MT, mimetics, agonists, antagonists, or inhibitors ofSAM-MT. The compositions may be administered alone or in combinationwith at least one other agent, such as stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0140] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0141] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0142] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0143] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0144] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0145] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0146] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Non-lipid polycationic amino polymers may also be used for delivery.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0147] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0148] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0149] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0150] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of SAM-MT, such labeling wouldinclude amount, frequency, and method of administration.

[0151] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0152] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0153] A therapeutically effective dose refers to that amount of activeingredient, for example SAM-MT or fragments thereof, antibodies ofSAM-MT, agonists, antagonists or inhibitors of SAM-MT, which amelioratesthe symptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the LD₅₀/ED₅₀ ratio.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0154] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0155] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

Diagnostics

[0156] In another embodiment, antibodies which specifically bind SAM-MTmay be used for the diagnosis of conditions or diseases characterized byexpression of SAM-MT, or in assays to monitor patients being treatedwith SAM-MT, agonists, antagonists or inhibitors. The antibodies usefulfor diagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for SAM-MT includemethods which utilize the antibody and a label to detect SAM-MT in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0157] A variety of protocols including ELISA, RIA, and FACS formeasuring SAM-MT are known in the art and provide a basis for diagnosingaltered or abnormal levels of SAM-MT expression. Normal or standardvalues for SAM-MT expression are established by combining body fluids orcell extracts taken from normal mammalian subjects, preferably human,with antibody to SAM-MT under conditions suitable for complex formation.The amount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of SAM-MTexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0158] In another embodiment of the invention, the polynucleotidesencoding SAM-MT may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof SAM-MT may be correlated with disease. The diagnostic assay may beused to distinguish between absence, presence, and excess expression ofSAM-MT, and to monitor regulation of SAM-MT levels during therapeuticintervention.

[0159] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding SAM-MT or closely related molecules, may be used to identifynucleic acid sequences which encode SAM-MT. The specificity of theprobe, whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding SAM-MT, alleles, or related sequences.

[0160] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the SAM-MT encoding sequences. The hybridization probes of thesubject invention may be DNA or RNA and derived from the nucleotidesequence of SEQ ID NO:2 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring SAM-MT.

[0161] Means for producing specific hybridization probes for DNAsencoding SAM-MT include the cloning of nucleic acid sequences encodingSAM-MT or SAM-MT derivatives into vectors for the production of mRNAprobes. Such vectors are known in the art, commercially available, andmay be used to synthesize RNA probes in vitro by means of the additionof the appropriate RNA polymerases and the appropriate labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as 32P or 35S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

[0162] Polynucleotide sequences encoding SAM-MT may be used for thediagnosis of conditions or disorders which are associated withexpression of SAM-MT. Examples of such conditions or disorders include:neoplastic disorders such as adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; immunological disorderssuch as AIDS, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjören's syndrome, Werner syndrome, and autoimmunethyroiditis; complications of cancer, hemodialysis, extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections and trauma; and vesicle trafficking disorders suchas cystic fibrosis, glucose-galactose malabsorption syndrome,hypercholesterolemia, diabetes insipidus, hyper- and hypoglycemia,goiter, Cushing's disease; gastrointestinal disorders including gastricand duodenal ulcers; and other conditions associated with abnormalvesicle trafficking such as allergies including hay fever and urticaria(hives); autoimmune hemolytic anemia; inflammatory bowel disease;Chediak-Higashi's syndrome; toxic shock syndrome; and traumatic tissuedamage. The polynucleotide sequences encoding SAM-MT may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dipstick, pin, ELISA assays ormicroarrays utilizing fluids or tissues from patient biopsies to detectaltered SAM-MT expression. Such qualitative or quantitative methods arewell known in the art.

[0163] In a particular aspect, the nucleotide sequences encoding SAM-MTmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding SAM-MT may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding SAM-MT in the sample indicates thepresence of the associated disease. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0164] In order to provide a basis for the diagnosis of diseaseassociated with expression of SAM-MT, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes SAM-MT,under conditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0165] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0166] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0167] Additional diagnostic uses for oligonucleotides designed from thesequences encoding SAM-MT may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5′->3′) and another with antisense (3′<-5′),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

[0168] Methods which may also be used to quantitate the expression ofSAM-MT include radiolabeling or biotinylating nucleotides,coamplification of a control nucleic acid, and standard curves ontowhich the experimental results are interpolated (Melby, P. C. et al.(1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.Biochem. 229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0169] In further embodiments, an oligonucleotide derived from any ofthe polynucleotide sequences described herein may be used as a target ina microarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

[0170] In one embodiment, the microarray is prepared and used accordingto the methods described in PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference.

[0171] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5′, or 3′, sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

[0172] In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

[0173] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0174] In order to conduct sample analysis using a microarray, the RNAor DNA from a biological sample is made into hybridization probes. ThemRNA is isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

[0175] In another embodiment of the invention, the nucleic acidsequences which encode SAM-MT may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome, to aspecific region of a chromosome or to artificial chromosomeconstructions, such as human artificial chromosomes (HACs), yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0176] Fluorescent in situ hybridization (FISH as described in Verma etal. (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.) may be correlated with other physical chromosomemapping techniques and genetic map data. Examples of genetic map datacan be found in various scientific journals or at Online MendelianInheritance in Man (OMIM). Correlation between the location of the geneencoding SAM-MT on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

[0177] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11 q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0178] In another embodiment of the invention, SAM-MT, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenSAM-MT and the agent being tested, may be measured.

[0179] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to SAM-MT largenumbers of different small test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with SAM-MT, or fragments thereof, and washed.Bound SAM-MT is then detected by methods well known in the art. PurifiedSAM-MT can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

[0180] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding SAM-MTspecifically compete with a test compound for binding SAM-MT. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with SAM-MT.

[0181] In additional embodiments, the nucleotide sequences which encodeSAM-MT may be used in any molecular biology techniques that have yet tobe developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

[0182] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0183] I THP1PLB01 cDNA Library Construction

[0184] THP-1 is a human leukemic cell line (ATCC TIB 202) derived fromthe blood of a one year-old boy with acute monocytic leukemia. Cellsused for the library were cultured for 48 hrs with 100 nM PMA diluted inDMSO and for 4 hrs with 1 μg/ml LPS. The cDNA libraries was customconstructed by Stratagene.

[0185] Stratagene prepared the cDNA library using a combination of oligod(T) and random priming. Double-stranded cDNA was blunted, ligated toEcoRI adaptors, digested with XhoI, size selected, and cloned into theXhoI and the EcoRI sites of the Lambda UNIZAP vector (Stratagene). Afterquality of the cDNA library was screened using DNA probes, thePBLUESCRIPT phagemid (Stratagene) was excised. Subsequently, thecustom-constructed library phage particles were infected into E. colihost strain XL1-BLUE (Stratagene).

[0186] II Isolation and Sequencing of cDNA Clones

[0187] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process, in which the host bacterial strain wasco-infected with both the library phage and an f1 helper phage. Thephagemid DNA was released from the cells, purified, and used to reinfectfresh host cells (SOLR, Stratagene) where double-stranded phagemid DNAwas produced. Plasmid DNA was released from the cells and purified usingthe REAL Prep 96 Plasmid Kit (Catalog #26173; QIAGEN, Inc). Therecommended protocol was employed except for the following changes: 1)the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog#22711, GIBCO BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%;2) the cultures were incubated for 19 hours after the wells wereinoculated and then lysed with 0.3 ml of lysis buffer; 3) followingisopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1ml of distilled water. After the last step in the protocol, samples weretransferred to a Beckman 96-well block for storage at 4° C.

[0188] The cDNAs were sequenced by the method of Sanger F. and A. R.Coulson (1975; J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno, Nev.) in combination with Peltier Thermal Cyclers(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems 377DNA Sequencing Systems; and the reading frame was determined.

[0189] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0190] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul, S. F. (1993) J.Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.215:403-410).

[0191] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R. F. and T. F. Smith(1992; Protein Engineering 5:35-51), incorporated herein by reference,can be used when dealing with primary sequence patterns and secondarystructure gap penalties. As disclosed in this application, the sequenceshave lengths of at least 49 nucleotides, and no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

[0192] The BLAST approach,as detailed in Karlin, S. and S. F. Atschul(1993; Proc. Nat. Acad. Sci. 90:5873-7) and incorporated herein byreference, searches for matches between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. In this application, threshold was set at10⁻²⁵ for nucleotides and 10⁻¹⁴ for peptides.

[0193] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology.

[0194] IV Northern Analysis

[0195] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0196] Analogous computer techniques using BLAST (Altschul, S. F. (1993)J. Mol. Evol. 36:290-300; Altschul, S. F. et al. (1990) J. Mol. Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0197] The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score 100

[0198] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0199] The results of northern analysis are reported as a list oflibraries in which the transcript encoding SAM-MT occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0200] V Extension of SAM-MT Encoding Polynucleotides

[0201] The nucleic acid sequence of the Incyte Clone 10625 was used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length. One primer was synthesized to initiateextension in the antisense direction, and the other was synthesized toextend sequence in the sense direction. Primers were used to facilitatethe extension of the known sequence “outward” generating ampliconscontaining new, unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06(National Biosciences), or another appropriate program, to be about 22to about 30 nucleotides in length, to have a GC content of 50% or more,and to anneal to the target sequence at temperatures of about 68° toabout 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

[0202] Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0203] High fidelity amplification was obtained by following theinstructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing theenzyme and reaction mix. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR wasperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (and holding)

[0204] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

[0205] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2×Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2×Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

[0206] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0207] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid, and sequenced.

[0208] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

[0209] VI Labeling and Use of Individual Hybridization Probes

[0210] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ³²P] adenosine triphosphate (Amersham)and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.). The labeledoligonucleotides are substantially purified with SEPHADEX G-25 superfineresin column (Pharmacia & Upjohn). A aliquot containing 10⁷ counts perminute of the labeled probe is used in a typical membrane-basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba I or Pvu II;DuPont NEN®).

[0211] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots areexposed in a PhosphorImager cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

[0212] VII Microarrays

[0213] To produce oligonucleotides for a microarray, the nucleotidesequence described herein is examined using a computer algorithm whichstarts at the 3′ end of the nucleotide sequence. The algorithmidentifies oligomers of defined length that are unique to the gene, havea GC content within a range suitable for hybridization, and lackpredicted secondary structure that would interfere with hybridization.The algorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/ WO95/11995, incorporated herein by reference).

[0214] In the alternative, a chemical coupling procedure and an ink jetdevice are used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a “gridded” array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

[0215] VIII Complementary Polynucleotides

[0216] Sequence complementary to the SAM-MT-encoding sequence, or anypart thereof, is used to decrease or inhibit expression of naturallyoccurring SAM-MT. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of SAM-MT, SEQ ID NO:1. To inhibit transcription, acomplementary oligonucleotide is designed from the most unique 5′sequence and used to prevent promoter binding to the coding sequence. Toinhibit translation, a complementary oligonucleotide is designed toprevent ribosomal binding to the SAM-MT-encoding transcript.

[0217] IX Expression of SAM-MT

[0218] Expression of SAM-MT is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express SAM-MT in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0219] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of SAM-MT into the bacterial growth media which can be useddirectly in the following assay for activity.

[0220] X Demonstration of SAM-MT Activity

[0221] A method that measures transfer of radiolabeled methyl groupsbetween a donor substrate and an acceptor substrate is used to determineSAM-MT activity (Bokar, J. A. et al. (supra)). Reaction mixtures (50 μlfinal volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mMdithiothreitol, 3% polyvinylalcohol, 1.5 μCi [methyl-³H]AdoMet (0.375 μMAdoMet) (DuPont-NEN), 0.6 μg SAM-MT, and acceptor substrate (0.4 μg[³⁵S]RNA or 6-mercaptopurine (6-MP) to 1 mM final concentration).Reaction mixtures are incubated at 30° C. for 30 minutes, then 65° C.for 5 minutes.

[0222] Analysis of [methyl-³H]RNA is as follows: 1) 50 μl of 2×loadingbuffer (20 mM tris-HCl, pH 7.6, 1 M LiCl, 1 mM EDTA, 1% sodium dodecylsulphate (SDS)) and 50 μl oligo d(T)-cellulose (10 mg/ml in 1×loadingbuffer) are added to the reaction mixture, and incubated at ambienttemperature with shaking for 30 minutes. 2) Reaction mixtures aretransferred to a 96-well filtration plate attached to a vacuumapparatus. 3) Each sample is washed sequentially with three 2.4 mlaliquots of 1×oligo d(T) loading buffer containing 0.5% SDS, 0.1% SDS,or no SDS. and 4) RNA is eluted with 300μl of water into a 96-wellcollection plate, transferred to scintillation vials containing liquidscintillant, and radioactivity determined.

[0223] Analysis of [methyl-³H]6-MP is as follows: 1) 500 μl 0.5 M boratebuffer, pH 10.0, and then 2.5 ml of 20% (v/v) isoamyl alcohol in tolueneare added to the reaction mixtures. 2) The samples are mixed by vigorousvortexing for ten seconds. 3) After centrifugation at 700 g for 10minutes, 1.5 ml of the organic phase is transferred to scintillationvials containing 0.5 ml absolute ethanol and liquid scintillant, andradioactivity determined. and 4) Results are corrected for theextraction of 6-MP into the organic phase (approximately 41%).

[0224] XI Production of SAM-MT Specific Antibodies

[0225] SAM-MT that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

[0226] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radio iodinated, goat anti-rabbit IgG.

[0227] XII Purification of Naturally Occurring SAM-MT Using SpecificAntibodies

[0228] Naturally occurring or recombinant SAM-MT is substantiallypurified by immunoaffinity chromatography using antibodies specific forSAM-MT. An immunoaffinity column is constructed by covalently couplingSAM-MT antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Pharmacia & Upjohn). After the coupling, theresin is blocked and washed according to the manufacturer'sinstructions.

[0229] Media containing SAM-MT is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of SAM-MT (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/SAM-MT binding (e.g., a buffer of pH 2-3 or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andSAM-MT is collected.

[0230] XIII Identification of Molecules Which Interact with SAM-MT

[0231] SAM-MT or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled SAM-MT, washed and any wells withlabeled SAM-MT complex are assayed. Data obtained using differentconcentrations of SAM-MT are used to calculate values for the number,affinity, and association of SAM-MT with the candidate molecules.

[0232] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 4 281 amino acids amino acid single linear THP1PLB01 10625 1 Met AlaSer Arg Gly Arg Arg Pro Glu His Gly Gly Pro Pro Glu Leu 1 5 10 15 PheTyr Asp Glu Thr Glu Ala Arg Lys Tyr Val Arg Asn Ser Arg Met 20 25 30 IleAsp Ile Gln Thr Arg Met Ala Gly Arg Ala Leu Glu Leu Leu Tyr 35 40 45 LeuPro Glu Asn Lys Pro Cys Tyr Leu Leu Asp Ile Gly Cys Gly Thr 50 55 60 GlyLeu Ser Gly Ser Tyr Leu Ser Asp Glu Gly His Tyr Trp Val Gly 65 70 75 80Leu Asp Ile Ser Pro Ala Met Leu Asp Glu Ala Val Asp Arg Glu Ile 85 90 95Glu Gly Asp Leu Leu Leu Gly Asp Met Gly Gln Gly Ile Pro Phe Lys 100 105110 Pro Gly Thr Phe Asp Gly Cys Ile Ser Ile Ser Ala Val Gln Trp Leu 115120 125 Cys Asn Ala Asn Lys Lys Ser Glu Asn Pro Ala Lys Arg Leu Tyr Cys130 135 140 Phe Phe Ala Ser Leu Phe Ser Val Leu Val Arg Gly Ser Arg AlaVal 145 150 155 160 Leu Gln Leu Tyr Pro Glu Asn Ser Glu Gln Leu Glu LeuIle Thr Thr 165 170 175 Gln Ala Thr Lys Ala Gly Phe Ser Gly Gly Met ValVal Asp Tyr Pro 180 185 190 Asn Ser Ala Lys Ala Lys Lys Phe Tyr Leu CysLeu Phe Ser Gly Pro 195 200 205 Ser Thr Phe Ile Pro Glu Gly Leu Ser GluAsn Gln Asp Glu Val Glu 210 215 220 Pro Arg Glu Ser Val Phe Thr Asn GluArg Phe Pro Leu Arg Met Ser 225 230 235 240 Arg Arg Gly Met Val Arg LysSer Arg Ala Trp Val Leu Glu Lys Lys 245 250 255 Glu Arg His Arg Arg GlnGly Arg Glu Val Arg Pro Asp Thr Gln Tyr 260 265 270 Thr Gly Arg Lys ArgLys Pro Arg Phe 275 280 1135 base pairs nucleic acid single linearTHP1PLB01 10625 2 AGTCGCAGGT GTGCTGCTGA GGCGTGAGAA TGGCGTCCCG CGGCCGGCGTCCGGAGCATG 60 GCGGACCCCC AGAGCTGTTT TATGACGAGA CAGAAGCCCG GAAATACGTTCGCAACTCAC 120 GGATGATTGA TATCCAGACC AGGATGGCTG GGCGAGCATT GGAGCTTCTTTATCTGCCAG 180 AGAATAAGCC CTGTTACCTG CTGGATATTG GCTGTGGCAC TGGGCTGAGTGGAAGTTATC 240 TGTCAGATGA AGGGCACTAT TGGGTGGGCC TGGATATCAG CCCTGCCATGCTGGATGAGG 300 CTGTGGACCG AGAGATAGAG GGAGACCTGC TGCTGGGGGA TATGGGCCAGGGCATCCCAT 360 TCAAGCCAGG CACATTTGAT GGTTGCATCA GCATTTCTGC TGTGCAGTGGCTCTGTAATG 420 CTAACAAGAA GTCTGAAAAC CCTGCCAAGC GCCTGTACTG CTTTTTTGCTTCTCTTTTTT 480 CTGTTCTCGT CCGGGGATCC CGAGCTGTCC TGCAGCTGTA CCCTGAGAACTCAGAGCAGT 540 TGGAGCTGAT CACAACCCAG GCCACAAAGG CAGGCTTCTC CGGTGGCATGGTGGTAGACT 600 ACCCTAACAG TGCCAAAGCA AAGAAATTCT ACCTCTGCTT GTTTTCTGGGCCTTCGACCT 660 TTATACCAGA GGGGCTGAGT GAAAATCAGG ATGAAGTTGA ACCCAGGGAGTCTGTGTTCA 720 CCAATGAGAG GTTCCCATTA AGGATGTCGA GGCGGGGAAT GGTGAGGAAGAGTCGGGCAT 780 GGGTGCTGGA GAAGAAGGAG CGGCACAGGC GCCAGGGCAG GGAAGTCAGACCTGACACCC 840 AGTACACCGG CCGCAAGCGC AAGCCCCGCT TCTAAGTCAC CACGCGGTTCTGGAAAGGCA 900 CTTGCCTCTG CACTTTTCTA TATTGTTCAG CTGACAAAGT AGTATTTTAGAAAAGTTCTA 960 AAGTTATAAA AATGTTTTCT GCAGTAAAAA AAAAGTTCTC TGGGCCGGGCGTGGTGGCTC 1020 ACACCTGTAA TCCCAGCACC TTGGGAGGCT GAGGTGGGAG GATCATTTGAGGCCAGGAGT 1080 TTGAGACCTG CCTGGGCAAC ATAATGAAAC TTCCTTTCCA GGGAGAAAAAAAAAA 1135 283 amino acids amino acid single linear GenBank 1065505 3Met Ala Ser Phe Lys Val Lys Pro Glu His Thr Gly Pro Pro Asp Leu 1 5 1015 Tyr Tyr Asn Glu Thr Glu Ala Ala Lys Tyr Ala Ser Asn Ser His Ile 20 2530 Thr Ala Ile Gln His Glu Met Ala Glu Arg Ala Leu Glu Leu Leu Ala 35 4045 Leu Pro Glu Gly Lys Ser Gly Phe Leu Leu Asp Ile Gly Cys Gly Thr 50 5560 Gly Met Ser Ser Glu Val Ile Leu Asp Ala Gly His Met Phe Val Gly 65 7075 80 Val Asp Val Ser Arg Pro Met Leu Glu Ile Ala Arg Gln Asp Glu Asp 8590 95 Leu Glu Ser Gly Asp Phe Ile His Gln Asp Met Gly Leu Gly Met Pro100 105 110 Phe Arg Pro Gly Ser Phe Asp Gly Ala Ile Ser Ile Ser Ala IleGln 115 120 125 Trp Leu Cys His Ala Asn Ala Ser Asp Glu Asn Pro Arg LysArg Leu 130 135 140 Leu Phe Phe Phe Gln Ser Leu Tyr Gly Cys Leu Gly ArgGly Ser Arg 145 150 155 160 Ala Val Phe Gln Phe Tyr Pro Glu Asn Asp GluGln Cys Asp Leu Ile 165 170 175 Met Gly Gln Ala His Lys Ala Gly Phe AsnGly Gly Leu Val Val Asp 180 185 190 Phe Pro Glu Ala Ala Lys Arg Lys LysVal Tyr Leu Val Leu Met Thr 195 200 205 Gly Gly Val Val Gln Leu Pro GlnAla Leu Thr Glu Asp Gly Glu Glu 210 215 220 Ser Arg Thr Gln Ile Asp AsnAla Gly Arg Arg Phe Val Trp Asn Ser 225 230 235 240 Arg Lys Asn Glu LysVal Ala Lys Gly Ser Lys Ala Trp Ile Glu Ala 245 250 255 Lys Arg Gln ArgGln Ile Lys Gln Gly Arg Asp Val Arg His Glu Ser 260 265 270 Lys Tyr SerGly Arg Lys Arg Lys Thr Lys Phe 275 280 275 amino acids amino acidsingle linear GenBank 1907189 4 Met Ser Arg Pro Glu Glu Leu Ala Pro ProGlu Ile Phe Tyr Asn Asp 1 5 10 15 Ser Glu Ala His Lys Tyr Thr Gly SerThr Arg Val Gln His Ile Gln 20 25 30 Ala Lys Met Thr Leu Arg Ala Leu GluLeu Leu Asn Leu Gln Pro Cys 35 40 45 Ser Phe Ile Leu Asp Ile Gly Cys GlySer Gly Leu Ser Gly Glu Ile 50 55 60 Leu Thr Gln Glu Gly Asp His Val TrpCys Gly Leu Asp Ile Ser Pro 65 70 75 80 Ser Met Leu Ala Thr Gly Leu SerArg Glu Leu Glu Gly Asp Leu Met 85 90 95 Leu Gln Asp Met Gly Thr Gly IlePro Phe Arg Ala Gly Ser Phe Asp 100 105 110 Ala Ala Ile Ser Ile Ser AlaIle Gln Trp Leu Cys Asn Ala Asp Thr 115 120 125 Ser Tyr Asn Asp Pro LysGln Arg Leu Met Arg Phe Phe Asn Thr Leu 130 135 140 Tyr Ala Ala Leu LysLys Gly Gly Lys Phe Val Ala Gln Phe Tyr Pro 145 150 155 160 Lys Asn AspAsp Gln Val Asp Asp Ile Leu Gln Ser Ala Lys Val Ala 165 170 175 Gly PheSer Gly Gly Leu Val Val Asp Asp Pro Glu Ser Lys Lys Asn 180 185 190 LysLys Tyr Tyr Leu Val Leu Ser Ser Gly Ala Pro Pro Gln Gly Glu 195 200 205Glu Gln Val Asn Leu Asp Gly Val Thr Met Asp Glu Glu Asn Val Asn 210 215220 Leu Lys Lys Gln Leu Arg Gln Arg Leu Lys Gly Gly Lys Asp Lys Glu 225230 235 240 Ser Ala Lys Ser Phe Ile Leu Arg Lys Lys Glu Leu Met Lys ArgArg 245 250 255 Gly Arg Lys Val Ala Lys Asp Ser Lys Phe Thr Gly Arg LysArg Arg 260 265 270 His Arg Phe 275

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO:1, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence of SEQ IDNO:1, c) a biologically active fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1, and d) an immunogenic fragment of apolypeptide having an amino acid sequence of SEQ ID NO:1.
 2. An isolatedpolypeptide of claim 1, having a sequence of SEQ ID NO:1.
 3. An isolatedpolynucleotide encoding a polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide of claim
 2. 5. An isolatedpolynucleotide of claim 4, having a sequence of SEQ ID NO:2.
 6. Arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 3. 7. A cell transformed with arecombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method forproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide has the sequence of SEQ ID NO:1.
 11. An isolatedantibody which specifically binds to a polypeptide of claim
 1. 12. Anisolated polynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence of SEQ ID NO:2, b) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence of SEQ ID NO:2, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional SAM-MT,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional SAM-MT, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional SAM-MT, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method for assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of SAM-MT in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of SAM-MT in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofSAM-MT in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide having an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibodies from said animal, and c) screening theisolated antibodies with the polypeptide, thereby identifying apolyclonal antibody which binds specifically to a polypeptide having anamino acid sequence of SEQ ID NO:1.
 37. An antibody produced by a methodof claim
 36. 38. A composition comprising the antibody of claim 37 and asuitable carrier.
 39. A method of making a monoclonal antibody with thespecificity of the antibody of claim 11, the method comprising: a)immunizing an animal with a polypeptide having an amino acid sequence ofSEQ ID NO:1, or an immunogenic fragment thereof, under conditions toelicit an antibody response, b) isolating antibody producing cells fromthe animal, c) fusing the antibody producing cells with immortalizedcells to form monoclonal antibody-producing hybridoma cells, d)culturing the hybridoma cells, and e) isolating from the culturemonoclonal antibody which binds specifically to a polypeptide having anamino acid sequence of SEQ ID NO:1.
 40. A monoclonal antibody producedby a method of claim
 39. 41. A composition comprising the antibody ofclaim 40 and a suitable carrier.
 42. The antibody of claim 11, whereinthe antibody is produced by screening a Fab expression library.
 43. Theantibody of claim 11, wherein the antibody is produced by screening arecombinant immunoglobulin library.
 44. A method of detecting apolypeptide having an amino acid sequence of SEQ ID NO:1 in a sample,the method comprising: a) incubating the antibody of claim 11 with asample under conditions to allow specific binding of the antibody andthe polypeptide, and b) detecting specific binding, wherein specificbinding indicates the presence of a polypeptide having an amino acidsequence of SEQ ID NO:1 in the sample.
 45. A method of purifying apolypeptide having an amino acid sequence of SEQ ID NO:1 from a sample,the method comprising: a) incubating the antibody of claim 11 with asample under conditions to allow specific binding of the antibody andthe polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:1.
 46. A microarray wherein at least one element of the microarrayis a polynucleotide of claim
 13. 47. A method of generating anexpression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.